Binding affinities of Schiff base Fe(II) complex with BSA and calf-thymus
DNA: Spectroscopic investigations and molecular docking analysis

Suparna Rudra, Somnath Dasmandal, Chiranjit Patra, Arjama Kundu,

Ambikesh Mahapatra

PII: S1386-1425(16)30229-3
DOI: doi: 10.1016/j.saa.2016.04.050
Reference: SAA 14413

To appear in:

Received date: 12 November 2015

Revised date: 26 March 2016
Accepted date: 27 April 2016

Please cite this article as: Suparna Rudra, Somnath Dasmandal, Chiranjit Patra, Ar-
jama Kundu, Ambikesh Mahapatra, Binding affinities of Schiff base Fe(II) complex with
BSA and calf-thymus DNA: Spectroscopic investigations and molecular docking analysis,
(2016), doi: 10.1016/j.saa.2016.04.050

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Binding affinities of Schiff base Fe(II) complex with BSA and calf-

thymus DNA : Spectroscopic investigations and molecular docking

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analysis

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Suparna Rudra, Somnath Dasmandal, Chiranjit Patra, Arjama Kundu and Ambikesh Mahapatra*

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Department of Chemistry, Jadavpur University, Kolkata 700 032, India

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Abstract

The binding interaction of a synthesized Schiff base Fe(II) complex with biological

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macromolecules viz., bovine serum albumin (BSA) and calf thymus(ct)-DNA have been

investigated using different spectroscopic techniques coupled with viscosity measurements at

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physiological pH and 298 K. Regular amendments in emission intensities of BSA upon the

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action of the complex indicate significant interaction between them, and the binding interaction

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have been characterized by Stern Volmer plots and thermodynamic binding parameters. On the

basis of this quenching technique one binding site with binding constant (Kb = (7.6±0.21) × 105)
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between complex and protein have been obtained at 298 K. Time-resolved fluorescence studies

have also been encountered to understand the mechanism of quenching induced by the complex.
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Binding affinities of the complex to the fluorophores of BSA namely tryptophan (Trp) and
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tyrosine (Tyr) have been judged by synchronous fluorescence studies. Secondary structural
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changes of BSA rooted by the complex has been revealed by CD spectra. On the other hand,
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hypochromicity of absorption spectra of the complex with the addition of ct-DNA and the

gradual reduction in emission intensities of ethidium bromide bound ct-DNA in presence of the
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complex indicate noticeable interaction between ct-DNA and the complex with the binding

constant (4.2 ± 0.11) × 106 M-1. Life-time measurements have been studied to determine the

relative amplitude of binding of the complex to ct-DNA base pairs. Mode of binding interaction

of the complex with ct-DNA has been deciphered by viscosity measurements. CD spectra have

also been used to understand the changes in ct-DNA structure upon binding with the metal

complex. Density functional theory (DFT) and molecular docking analysis have been employed

in highlighting the interactive phenomenon and binding location of the complex with the

macromolecules.
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Keywords: Schiff base, Fe(II) complex, serum albumin, ct-DNA

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1. Introduction

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The recent decade has witnessed a phenomenal rise in the growth of Schiff base metal complexes

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due to their preparative accessibilities, structural varieties (co-ordination number, geometry) and

variable oxidation state [1]. The metal complexes with chelating ligands containing O, N, and S

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as donors show evergreen growing curiosity owing to their applications in biochemical reactions
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and biological regulators [2, 3]. However, transition metal ions are known to enhance the

biological activity of different Schiff base ligands [4]. Many of such transition metal complexes
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are recently being employed as models of antibacterial, antifungal, anti-inflammatory and

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anticancer drugs. This positively charged metal complexes show tremendous affinity towards the

negatively charged bio-molecules like ATP, proteins, nucleic acids etc under physiological
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conditions [5(a), (b)]. Therefore, the preparation of such metal complexes draws significant

interests to the recent researchers [6].

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Serum albumins are the most abundant proteins in blood plasma, which carries various

endogenous and exogenous compounds containing amino acids, fatty acids, hormones, metals

and chemical constituents [7]. They maintain the blood pH and also contribute about 80% to the

osmotic blood pressure [8]. In addition with binding affinities they can increase the solubility of

drugs in blood plasma. BSA is an important transport protein having 583 amino acid residues in

its single polypeptide chain with molar mass of 66,000 Da [9]. It contains two tryptophan (Trp)

residues that are housed at positions 134 and 213 in the amino acid sequence of the protein. The

primary structure of the protein also consists of nine loops which are held together by 17
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disulfide bonds. These bonds permit the protein to be flexible for structural changes induced by

different experimental conditions [10].

DNA is one of the basic genetic materials of life [11]. In modern era metal toxicity on nucleic

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acids has developed a great interest to the researchers. The binding mode of metal complexes to

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DNA double helical structure exists in three different ways viz. coulombic interaction, groove

binding and intercalative interaction [12]. The intercalative binding of metal complexes with

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DNA can show antitumor activity by inhibition of DNA replication in rapidly mounting cancer

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cells. It is reported in literature that without forming of covalent bond, some intercalaters

intercalate in-between the base pairs of DNA by overlapping of pi-electrons [13].

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8-amino quinoline is a widely used constituent in the preparation of Schiff bases and subsequent

synthesis of drug molecules that play significant role in physiological functions [14]. Quinoline
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and its derivatives exhibit extensive π-staking ability and coordination property which makes
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them biologically important [15].

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In our present work, we intend to study the type of binding interaction of a Fe(II) complex
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([Fe(pmaq)2]2+) (Scheme 1), engineered by a synthesized Schiff base (2-pyridinylmethylene-8-

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quinolinyl, designated as ‘pmaq’) comprising of 8-amino quinoline & pyridine-2-

carboxaldehyde, with bio-molecules namely BSA and ct-DNA. Binding of the complex with bio-

molecules may induce considerable changes in the conformation of BSA and ct-DNA. The

interactive phenomenon between the complex and the macromolecules has been exploited by

absorption, fluorescence and circular dichroism (CD) spectra under the physiological conditions.

Viscometric technique has been employed for understanding the type of binding of the complex

with ct-DNA. A quantum mechanical approach from density functional theory (DFT) and

molecular docking analysis have been attempted to support the experimental findings.
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Scheme 1 Structure of [Fe(pmaq)2]2+ complex

2. Materials and methods

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2.1. Materials
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8-amino quinoline, pyridine-2-carboxaldehyde, ferrous sulphate, bovine serum albumin (BSA),

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calf thymus- DNA (ct-DNA), ethidium bromide (EB) have been purchased from Sigma Aldrich
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(USA) and used as received. All other chemicals have been procured from the commercial
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sources as per requirements and used after purification. All the sample solutions have been
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prepared in 10 mM tris- HCl/ 10 mM NaCl buffer (pH 7.4) otherwise mentioned. The buffer

solution has been made in deionised ultrapure water from Millipore Synergy System (Merck,

India) using tris base that has been purchased from Merck, India. The absorbance of the buffered

ct-DNA solution at 260 nm and 280 nm shows a ratio of 1.8 – 1.9 indicating that ct-DNA is pure

and sufficiently free from protein [16]. The concentration of ct-DNA has been determined by

measuring absorbance at 260 nm using the molar extinction coefficient(ε) value of 6600 M-1 cm-1

[17]. BSA stock solution has also been prepared in 10 mM tris- HCl buffer measuring the
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absorbance at λmax of 280 nm with ε value of 43,824 M-1 cm-1[18]. The stock solutions of BSA

and ct-DNA have been stored in the dark at 4 ○C and used within 4 days.

2.2. Methods

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2.2.1. Experimental

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Electronic spectra have been recorded by using Shimadzu RF-5000 spectrophotometer with

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quartz cells from Hellma having 1 cm path length. The fluorometric analysis has been carried out

employing Shimadzu RF-5301PC spectrofluorimeter (Kyoto, Japan). For fluorescence lifetime

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measurements, photo excitation has been done using a pico second diode laser (IBH nanoled 07).
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The data have been collected by using Hamanatsu MCP photomultiplier (R3809) and analyzed

by IBH DAS-6 software. Viscometric measurements have been carried out by a Cannon –
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Manning semi-micro dilution viscometer type-75 which is placed vertically at constant

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temperature bath. Circular Dichroism (CD) spectra have been recorded using PC-driven JASCO
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spectropolarimeter (Japan) equipped with a Peltier temperature controller and thermal

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programmer (PED-425L/15). 1H NMR (300 MHz) has been recorded from a Bruker (AC) 300

MHz FT-NMR spectrometer using TMS as an internal standard. A FT-IR spectrum (KBr disk,
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4000 – 400 cm-1) has been recorded from a Perkin Elmer LX-1 FTIR spectrophotometer. ESI-

MS+ (m/z) of the complex has been recorded on HR MS spectrophotometer (Model: QTOF

Micro YA263).

2.2.1.1. Preparation of [Fe(pmaq)2]2+ complex

Synthesis of ligand, pmaq

The Schiff base ligand has been synthesized following the reported procedure [19]. Hot ethanolic

solution of 8-amino quinoline (1.44 g, 1.0 mmol) has been mixed with ethanolic solution of

pyridine-2-carboxaldehyde (0.95 mL, 1.0 mM). The mixture is then refluxed at 80 ○C for 5 h
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with constant stirring. After cooling it is then washed with diethyl ether and dried in oven. The

characterization of the synthesized ligand has been performed using UV-Vis spectroscopy and

NMR spectroscopy which has been shown in Fig S1 (a) (S stands for supporting information)

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[19].

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Synthesis of the complex

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For the preparation of the complex, alcoholic solution of 0.5 mmol of FeSO4•6H2O has been

treated with 1.0 mmol alcoholic solution of the synthesized ligand. The mixture is heated to

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70.○C with continuous stirring. A dark green precipitate is obtained which is filtered & washed
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with diethyl ether and dried in desiccators. The complex has been then re-crystallized using

water-ethanol mixture and finally characterized by UV-Vis, IR and Mass spectroscopy [19]. The

representative IR spectrum {  (CN) = 1639.16 cm-1,  (py/qu) = 615.38 cm-1} has been shown
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in Fig S1 (b) .The characteristic mass spectrum has been shown in Fig S1 (c) ESI-MS (M++H),

261.21.
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2.2.1.2. BSA binding experiment

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The binding interaction between BSA and Fe(II) complex has been deciphered employing

steady-state and time-resolved fluorescence measurements. The fluorescence spectra of buffered

BSA solution have been recorded in the wavelength region of 290 - 460 nm using excitation/

emission slit width of 3 / 3 nm. The BSA solution of concentration 3.0 μM has been used in the

fluorometric titration. The entire experiment has been carried out within 1% dilution of the

buffered BSA solution. Circular Dichroism (CD) spectra of BSA have also been recorded in the

presence and absence of complex for better understanding of the BSA-complex interactive

phenomenon. For CD measurements, the concentration of BSA has been kept at 0.3 μM due to
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instrumental requirements. The CD results have been expressed in terms of mean residue

ellipticity (MRE) in deg cm2 dmol-1 using eq. 1 [20].

MRE = θobs /10 n l [P] (1)

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Where θobs is the CD in millidegree, n is the number of amino acid residue (583 for BSA), l is the

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path length of the cell, P is the molar concentration of the albumin.

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2.2.1.3. DNA binding experiment

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Absorption studies

UV-Vis absorption spectroscopy has been employed to investigate the interaction between ct-
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DNA and metal complex. Quartz cells form Hellma of 1 cm path length has been used in this

study. The buffered solution of ct-DNA has been added successively to a fixed amount of the
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complex. During the optical titration of the complex, an equal amount of buffered ct-DNA

solution has been added to both the sample and reference cells. The mixture containing metal
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complex and the added ct-DNA has been incubated each time for 5 minute prior to record
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spectrum.
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Fluorometric studies

The relative binding capacity of the metal complex to ct-DNA has been examined from steady-

state and time-resolved fluorescence studies using ethidium bromide (EB) as fluorescence probe.

In steady-state fluorescence, the EB bound ct-DNA has been excited at 510 nm, and the emission

spectra have been recorded in the wavelength region of 540 – 750 nm with excitation/ emission

slit width of 3 / 5 nm. The complex solution is allowed to add gradually to the buffered EB

bound ct-DNA and each addition has been carried out after incubating the mixture for 5 minute.

This experiment has been carried out within 1.5% dilution of the buffered ct-DNA solution.
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Viscosity measurement

Viscometric studies have been performed using 5.0 × 10-4 M ct-DNA solution. A 700 µL of

buffered ct-DNA solution has been placed in the viscometer and the complex solution has been

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directly added to it. The measurements have been done from the flow time of ct-DNA solution in

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the absence and presence of the metal complex. Each measurements has been repeated at least

thrice and the reproducibility of the data has been estimated to be within ± 0.01 s. Relative

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specific viscosity has been calculated by using the eq. 2 [21]:

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ηsp [tcomplete  t0 ]
 (2)
spMA [tcontrol  t0 ]

where ηsp and  sp are the specific viscosity of ct-DNA in the absence and presence of complex

respectively, tcomplete and tcontrol are the respective flow time of complex and control solution and
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t0 is the flow time of free buffer solution.

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Circular Dichorism studies

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CD spectra have been recorded in the wavelength range of 220 - 330 nm with scan speed of 200

nm min-1. An average of three scans has been done for each spectrum at 298 K. The base line has
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been subtracted from each data set.

2.2.2. Theoretical

For docking studies, the ground state geometry of the Schiff base metal complex has been

optimized employing density functional theory (DFT) in conjugation with B3LYP functional and

6-31G* standard basis set in Gaussian 09 program suit [22]. The required pdb structure of the

complex has been derived from the output file of the optimized structure. The crystal structures

of BSA and ct-DNA used in the docking studies have been obtained from Protein Data Bank

(PDB) with pdb id: 4F5S (BSA) and 1BNA (ct-DNA). Polar hydrogen atoms and Gasteiger
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charges have been added then to prepare them for docking. The metal complex has been

considered as ligand and the bio-molecule (BSA and ct-DNA) as receptor for docking studies.

The molecular docking analysis have been carried out applying the Lamarckian Genetic

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Algorithm (LGA) implemented in AutoDock 4.2 [23]. BSA and ct-DNA have been laid over a

three dimensional grid box with grid volume of (126 × 126 × 126) Å3 along x-, y-, and z- axis

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with 0.675 Å spacing. The output from AutoDock has been further analyzed using PyMOL

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software [24].

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3. Results and Discussion

3.1. BSA–[Fe(pmaq)2]2+ complex binding studies

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3.1.1. Steady-state fluorescence studies

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Fluorometric technique achieves wide spread application in investigating of interactions between

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metal complex and protein molecules [25]. The intrinsic fluorescence of BSA is mainly

dependent on two aromatic amino acids namely tryptophan (Trp) and tyrosine (Tyr). BSA
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contains three homologous α-helical domains (I, II & III) each with two sub-domains A & B.

BSA possesses 20 tyrosine residues in its different domains with high abundances in domain I

[26]. It also contains two tryptophan residues, namely Trp-134 and Trp-213. The Trp-134 is

located in domain I (sub-domain IB) and exposed towards the surface of the protein molecule,

whereas Trp-213 presents in a hydrophobic cavity of the protein in domain II (sub-domain IIA)

[7, 27]. Thus in our present work, the interaction between the metal complex and BSA have been

monitored upon exciting the protein at 280 nm where both Trp and Tyr residues get excited. The
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variations of emission spectra of buffered BSA (3.0 µM) upon successive addition of metal

complex have been shown in Fig. 1.

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Fig. 1. Variation of fluorescence spectra of BSA in absence and presence of metal complex (λex =
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280 nm) at 298 K, where (i) → (vii) corresponds to 0.0, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 µM Fe(II)
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complex solution. [BSA]0 = 3.0 µM.

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The Fig. 1 shows a regular reduction in the fluorescence intensity of BSA up to 6.0 µM of the

complex concomitant with a 2 nm blue shift of the emission maximum. Under the experimental

condition, the complex does not produce any emission in the specified range of study (290 - 460

nm) implying that the emission spectra as we observe in Fig. 1 are solely contributed by the

fluorophores of BSA [28]. The blue shift of emission maxima clearly indicates the increment of

hydrophobicity around the fluorophores [10]. However, the gradual decrease in the fluorescence

intensity of BSA suggests that the protein structure gets unfolded upon the action of the complex.
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The accessibility of the protein to the complex has been observed by fluorescence quenching

experiment using Stern-Volmer equation [25].

F0
 1  K sv [Q] (3)

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F

where F0 and F are steady-state fluorescence intensities of BSA in the absence and presence of

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quencher (here, [Fe(pmaq)2]2+), τ0 is average life-time in the absence of quencher, kq is

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bimolecular quenching rate constant and KSV (≡ kqτ0) is Stern-Volmer constant. The Stern-

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Volmer plot (Inset of Fig. 1) for the binding of metal complex with BSA shows upward

curvature towards Y axis. The nonlinearity of the curve implies the presence of both static and
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dynamic quenching by the same quencher [29]. Therefore, modified Stern- Volmer equation has

been used to analyze the quenching data [30].

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F0  1  1  1
  
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(4)
( F0  F )  f a K a   Q   f a
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Where fa is the fraction of accessible fluorescence and Ka is the effective quenching constant.
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From the plot of F0/ (F0-F) versus 1/[Q]0 (Fig. S2), Ka and fa have been obtained to be
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(2.43±0.06) × 106 M-1 and 0.63 from the slope and intercept of the straight line respectively. For

better understanding of the quenching mechanism, time-resolved fluorescence measurements

have been performed as discussed in the following section.

3.1.2. Time-resolved fluorescence measurements

Time-resolved fluorescence studies bear exigent information about the quenching mechanism of

various additives in protein microenvironment [7]. In our present protein-metal complex system,

the gradual amendments in fluorescence life-time (τ) of Trp residues upon the continuous

addition of metal complex indicates the dynamic nature of the quenching mechanism.
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Fig. 2. Fluorescence life-time decay of BSA in the absence and presence of metal complex at 298
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K, [BSA]0 = 3.0 µM.
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The representative life-time decay profiles of Trp in the absence and presence of metal complex

have been shown in Fig. 2. It is very difficult to assign the individual component of life-time in a
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multi-exponential decay. Despite of the fact we have made an attempt to interpret the relative
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amplitudes of life-time of Trp in the absence and presence of metal complex. The Trp emission
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has been fitted to a bi-exponential decay and the resultant parameters have been represented in

Table 1. A recent study by Mukherjee et al revealed that the fluorescence life-time of BSA is

contributed by three rotational conformers of Trp, among which two are rapidly inter-converting

(say A & B), and the third one (say C) is rather stable [31]. These rotamers of Trp residues have

been depicted in Scheme 2. The shorter life-time (τ1) i.e., 4.12 ns has been assigned to conformer

C, while the longer life-time (τ2), 7.32 ns, is due to the rapidly inter-converting A and B

conformers. From Table 1, we can observe that the contribution from C conformer changes from
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about 41.36% to 52.34%, and that for A (and/ or B) conformers from about 58.64 % to 47.66%

at 10 μM of the complex.

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H NH3
N
H H
NH3

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H H CO2

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H CO2

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H
HN
(A) (B)
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NH3 NH
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H CO2
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(C)
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Scheme 2. Rotational conformers of Trp residues

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Table 1. Fluorescence decay parameters of BSA in the absence and presence of Fe(II) complex

[Fe(pmaq)2]0 1 2  
A1 A2 χ2
µM (ns) (ns) (ns)

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0 4.12 0.4136 7.32 0.5864 6.03 1.04

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1.15 3.85 0.4250 7.10 0.5750 5.72 1.07

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3.5 2.98 0.4410 6.98 0.5590 5.22 1.05

5.5 2.76 0.4650 6.78 0.5350 4.91 1.04

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8.0 2.53 0.4939 6.14 0.5061 4.34 1.06

10.0 2.19 0.5234

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The average fluorescence life-time of BSA in the absence (‹τ0›) and presence (‹τ›) of metal
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complex have been used to estimate the dynamic quenching constant (KD) employing the
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following Eq. 5 [29].

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 0 
 1  K D [Q] (5)
 
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From the slope of the plot of (‹τ0›/‹τ›)-1 versus [Q] (Fig. S3), KD has been obtained to be

(4.46±0.12)× 104 M-1. Moreover, the static quenching constant, KS can also be evaluated using

KD value according to Eq. 6 [30].

 F0  F 
 
 F   ( K  K )  K K [Q]
s D S D (6)
[Q]
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The value of KS as calculated from the slope of the plot {(F0-F/F)/ [Q]0} versus [Q]0 (Fig. S4) is

found as (4.6±0.11) × 105 M-1. The results indicate that the quenching mechanism is

predominantly static in nature.

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3.1.3. Determination of binding parameters

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The binding parameters for BSA-[Fe(pmaq)2]2+ complexation has been estimated employing a

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double-logarithmic equation as follows [32]

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F F   
 Kb  
 Q  

log  0   log  n   n log   (7)
 F  M 

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 M  

Where Kb and n are designated as the binding constant and the number of binding sites
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respectively. According to Eq. 7, a plot of log [(F0-F)/F] versus log([Q]0/M) produce a straight
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line out of which the values of Kb and n have been calculated from the intercept and slope
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respectively (Fig. 3). The estimated binding parameters at different experimental temperature
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have been presented in Table 2. At room temperature i.e., 298 K, the value of n is found to be

very close to 1 implying the presence of one binding site for [Fe(pmaq)2]2+ in BSA. With the
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progressive rise of temperature, the value of both the binding constant and the number binding

site increases which suggests that the BSA molecule may open a further binding site to bind to

another metal complex [25].

When we consider the temperature dependence on the fluorescence quenching process, we may

think about the Arrhenius theory. According to this theory rate constant of a reaction increases

with increase of temperature. For classical static quenching, the rate constant decreases as we

raise the temperature, mainly due to instability of the BSA–[Fe(pmaq)2]2+ complex, but the

reverse is happened for the dynamic quenching, due to increment of excited state complexation.
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In our present study, although the static quenching process is more effective than the dynamic

quenching, the rate constant increases with temperature. This can be ascribed to the predominant

effect of temperature on the rate acceleration over the tendency to induce instability in the BSA–

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[Fe(pmaq)2]2+ complex that leads to decrease the rate.

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Fig. 3. Plot of log[(F0-F)/F] versus log([Q]0/M) at different temperatures representing effect of

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temperature on binding of complex with BSA, pH 7.4.

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Table 2. Various binding and thermodynamic parameters of BSA – [Fe(pmaq)2]2+ system at

different temperatures.

ΔG° ΔH° ΔS°

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T (K) n (kJ mol-1) (kJ mol-1) (J mol-1 K-1)

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293 0.95±0.01 − 30.18±0.72 567.71±14.75

− 33.55±0.81

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298 1.06±0.02 196.52±5.82 546.88±13.88

303 1.17±0.01 − 37.24±0.95 525.68±13.66

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308 1.29±0.02 − 41.75±0.98 502.50±13.56
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3.1.4. Determination of thermodynamic parameters

The thermodynamic parameters for the binding of drug molecule with biological
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macromolecules such as serum protein often provide valuable information in predicting the mode

of interaction between them. A number of possible interactions in this regard are: coulombic,
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hydrophobic and van der Waals interaction [33]. In our present study, the values of binding

constant have been determined at four different temperatures and shown in Table 2. The
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thermodynamic parameters viz., standard enthalpy change (ΔH°) and standard entropy change

(ΔS°) have been estimated using van’t Hoff equation [25]:

H S
ln Kb    (8)
RT R

The linearity of the plot of RlnKb versus 1/T indicates no significant change of enthalpy within

the specified temperature range (Fig. S5). The values of ΔH° and ΔS° have been obtained from

the slope and the intercept of the plot respectively. A positive value of ΔH° i.e., 196.52±5.82 kJ
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mol-1 indicates that the binding process is endothermic in nature and the driving force is

hydrophobic interaction rather than coulombic and van der Waals forces [25].

The standard free energy change for the binding process has been evaluated from Eq. 9 as

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G  H  T S   RT ln Kb

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(9)

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The negative value of ΔG° at constant pressure and temperature (-33.55±0.81 kJ mol-1) reveals

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the spontaneity of the reaction. Using this equation the entropy changes also have been

calculated at different temperatures which are shown at Table 1.

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3.1.5. Synchronous fluorescence studies
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Synchronous fluorescence spectra (SFS) store significant information while investigating multi-
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component system [34]. SFS can also be used to explore the protein-complex interactive
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phenomenon by applying wavelength interval (Δλ = λexcitation – λemission) at 15 or 60 nm which

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serves the characteristic information about the environment of tryptophan and tyrosine residues

respectively [7, 10]. The synchronous fluorescence spectra of BSA in the presence of metal
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complex have been shown in Fig. 4.

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Fig. 4. Synchronous fluorescence spectra (a) Δλ = 15 nm, (b) Δλ = 60 nm of BSA by

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[Fe(pmaq)2]2+ complex with different concentrations (0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 µM). The

arrow indicates the decrease of fluorescence intensity with increasing Fe(II) complex

concentration.

At Δλ = 15 nm (Fig. 4a), no shift in the wavelength of maxima is observed upon the addition of

metal complex to BSA indicating that the microenvironment around Tyr residues remain the

same as that in native BSA. While the maximum emission wavelength for tryptophan (Δλ = 60

nm) is slightly blue shifted (Fig. 4b). This clearly indicates that the polarity of the
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microenvironment around Trp residue decreases in the presence of metal complex. Therefore, the

SFS studies reveal that the metal complex binds to BSA near Trp residue.

3.1.6. Circular Dichroism studies

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CD is a well-established and sensitive tool for the analysis of protein secondary structure and

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hence useful for monitoring the conformational changes of protein upon the interaction of metal

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complexes [32]. In the CD spectrum, BSA exhibits two negative bands at 208 nm and 222 nm

originating from π→π* and n→transition of the peptide bond of α-helix respectively [34, 35].

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Fig. 5 shows the far-UV CD spectra of BSA in the absence and presence of the metal complex
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revealing that the binding of metal complex to BSA results in a decrease of band intensity

without any significant shift of the peak. Any change of α-helical structure of BSA is used to
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ascribe qualitatively by the amendment in the ellipticity at 222 nm (–θ222) [36]. The content of α-
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helix of BSA in its native state and the presence of complex have been calculated employing a
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computer based programmed, CDNN analysis. From the quantitative analysis it is observed that
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the α-helicity of BSA decreases from 59.4% (native state) to 56.8% at 10 µM complex
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concentration. In our present work, the CD measurements have been performed using 0.3 µM

BSA (as mentioned in the experimental section), whereas the concentration of metal complex has

been kept the same to that used in the previous experiments. Thus there will hardly be any such

correspondence to the data obtained from CD spectra and that from previous techniques. The

diminution in the α-helical content of BSA upon the action of complex implies the loss of

biological activity of the protein. This is because; the biological activity of protein is associated

with its secondary structural contents. The CD results reveal that the metal complex binds with

amino acid residues of the main polypeptide chain thereby destabilizing the protein structure

[37].
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Fig. 5. CD spectra of BSA in absence and presence of metal complex in tris HCl buffer (pH 7.4),
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[BSA]0 = 0.3 µM.
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3.2. ct-DNA–[Fe(pmaq)2]2+ complex binding studies

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3.2.1. Absorption studies

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Absorption spectroscopy is the most commonly employed technique for examining the
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interaction of metal complexes with ct-DNA [38]. The complex, [Fe(pmaq)2]2+ exhibits an

absorption spectra in the visible region with a maximum at 660 nm. Fig. 6 shows the gradual

reduction of absorbance of the complex upon successive addition of ct-DNA indicating

significant binding interaction to be present between them. The minor bathochromic shift of the

absorption maximum by 1 nm suggests that the [Fe(pmaq)2]2+ complex interacts with ct-DNA

most likely through a partial intercalative and / or a minor groove binding mode [39].
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Fig. 6. Absorption spectra of the [Fe(pmaq)2]2+ complex in absence and presence of ct-DNA

solution. Curves (i) → (vi) correspond to (0.0, 12.5, 25.0, 37.5, 50.0 and 62.5 μM) of ct-DNA
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concentration, [Complex]0 = 30.0 μM.

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3.2.2. Steady-state fluorescence studies

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The fluorescence property has not been observed for the [Fe(pmaq)2]2+ complex at room
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temperature in the absence or the presence of ct-DNA. Thus in order to clarify the mode of

binding interaction of the metal complex with ct-DNA, it is customary to use ethidium bromide

(EB) as a probe [1]. EB, a planar cationic dye, shows intense fluorescence after intercalating

between adjacent base pairs in the ct-DNA double helix [11]. The enhanced fluorescence of EB-

ct-DNA adduct can be quenched by quencher molecule which may be due to the release of EB or

the breaking of ct-DNA structure [40].

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Fig. 7. Variation of fluorescence emission spectra of EB bound ct-DNA in absence and presence
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of [Fe(pmaq)2]2+complex, [EB]0 = 5.0 µM, [DNA]0 = 5.0 µM, λex = 510 nm. Curves (i) → (xiv)
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correspond to 0.0, 1.8, 3.6, 5.4, 7.2, 9.0, 10.8, 12.6, 14.4, 16.2, 18.0, 19.8, 21.6, 23.4 µM Fe(II)
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complex solution.
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In our present work, Fig. 7 shows an appreciable reduction in the emission intensity of EB bound

ct-DNA upon the addition metal complex which gives a measure of the binding affinity of the

complex and stacking interaction between the adjacent base pairs of ct-DNA. The binding

strength of the complex with ct-DNA have been understood from classical Stern-Volmer

equation [41]

I0
 1  K sq r (10)
I
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where, I0 and I are fluorescence intensities in the absence and presence of the complex, Ksq is the

linear Stern-Volmer quenching constant and r is denoted as the ratio of total concentration of the

complex to that of the ct-DNA i.e., [complex]0/[ct-DNA]0. The value of Ksq has been obtained

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from the slope of the linear plot (S6) and found as 0.58, which is almost similar with that of

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some reported transition metal complexes (Ksq = 0.41 and 0.53) [42]. The result indicates partial

intercalation of the [Fe(pmaq)2]2+ complex between ct-DNA base pairs, and being a planer

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system the Schiff base ligand used in our study plays an important role for the intercalation of

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the complex with ct-DNA [43].
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Fig.8. A plot of log[(F0-F)/F] against log{[Q]0/M} at 298 K for EB bound ct-DNA with varying

the Fe(II) complex concentration from 1.8 to 23.4 µM at pH 7.4.

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The evaluation of binding constant (Kb) and stoichiometry of binding (n) is often helpful in

characterizing the binding interaction. In this perspective, we use the emission data employing eq

7 (same as the case for BSA), and the plot of log[(F0-F)/F] versus log([Q]0/M) is found to

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produce a straight line from which Kb and n can be calculated from the intercept and slope

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respectively (Fig. 8). The value of n is found to be very close to 1 indicating the binding of metal

complex to ct-DNA in an independent binding site. The evaluated value of Kb ((4.2 ±0.11)× 106

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M-1) suggests a moderately strong binding of the complex to ct-DNA.

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The relative binding constant of the complex can be obtained from Fig. 7 using the following

equation
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KEB × [EB]1/2 = Kapp × [complex]1/2 (11)
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where [EB]1/2 (5.0 µM) and [complex]1/2 are the concentration of the EB and the complex
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respectively at 50% reduction of the fluorescence intensity, KEB is the binding constant between
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EB and ct-DNA and the value of which is taken as 1.0 × 107 M-1 [12]. Using these values, the
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apparent ct-DNA binding constant (Kapp) for the complex is calculated as (4.63±0.13) × 106 M-1.
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The value of Kapp implies that the complex binds to ct-DNA in a considerably stronger way, but

less stronger than the classical intercalater EB. Hence, partial intercalation and/ or groove

binding of the complex to ct-DNA can easily be anticipated.

The binding of the metal complex with ct-DNA has been observed from the UV-Vis

spectroscopy which indicates the ground state complex formation between them. This indicates

the presence of static quenching. But in order to clarify the presence of dynamic quenching time

dependent Fluorometric studies have been done.

3.2.3. Time-resolved fluorescence measurements

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Time-resolved fluorescence spectroscopy is a sensitive technique for the investigation of

conformational changes of DNA both in chemical and cellular systems resulting from its

interaction with intercalating and/or groove binding drug molecules [44]. In our present study,

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the fluorescence decay profile of EB bound ct-DNA in the absence and the presence of metal

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complex are shown in Fig. 9. From Fig. 9, it is clear that the life-time of EB–ct-DNA system

reduces upon the action of the metal complex implying the dynamic contribution to the overall

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quenching mechanism along with the presence of ground state interaction which we have already

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observed. The emission of EB–ct-DNA adduct has been fitted to a biexponential decay, and the

deconvoluted data are represented in Table 3. In the absence of metal complex, the two
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components contributed to the average life-time (‹τ›) of EB–ct-DNA system may be ascribed to

free EB (τ1) and EB–ct-DNA adduct (τ2) [45]. The shorter life-time (τ1), 2.36 ns, has been
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assigned to free EB; while the longer life-time (τ2), 20.98 ns, is due to the intercalated EB [46].

At this stage, we have made an effort to explain the relative amplitude of the two components in
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the excited state upon gradual addition of the complex to EB–ct-DNA system. From Table 3, it is

observed that the contribution of EB–ct-DNA adduct changes from about 65.5 to 1.3%, whereas
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that of free EB changes from about 35.5 to 98.7% at 40.0 µM of [Fe(pmaq)2]2+. These are

attributed to the fact that the [Fe(pmaq)2]2+ complex displaces the intercalated EB from ct-DNA

base pairs and subsequently releases EB to the bulk aqueous phase. At the maximum added

concentration of the complex, the average life-time (‹τ›) of EB–ct-DNA system reduces to 1.82

ns (Table 3) which is very much close to the life-time of free EB i.e., 1.67 ns as reported by

Greenstock et al [45]. Hence, the life-time values suggest strong binding of the complex to ct-

DNA, and the binding may be through intercalative mode.

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Fig. 9. Fluorescence decay curve of EB bound ct-DNA ([DNA]0 = 25 µM) in absence and
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presence of [Fe(pmaq)2]2+ complex, where, (i) → (ix) correspond to 0.0, 5.0, 10.0, 15.0, 20.0,
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25.0, 30.0, 35.0 and 40.0 µM Fe(II) complex solution.

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Table 3. Fluorescence lifetime of EB bound ct-DNA in absence and presence of [Fe(pmaq)2]2+

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complex.
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[Fe(pmaq)2 1 2  
2
A1 A2 χ2
] µM (ns) (ns) (ns)
0.0 2.3556 0.3543 20.9814 0.6457 14.3 1.053

5.0 2.2458 0.5783 18.7360 0.4217 9.2 1.001

10.0 2.1608 0.7343 17.0971 0.2657 6.13 0.961

15.0 1.9451 0.8596 15.1295 0.1404 3.82 1.031

20.0 1.8880 0.8997 14.0392 0.1003 3.12 1.028

25.0 1.8063 0.9321 12.7467 0.0679 2.55 1.024

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30.0 1.7887 0.9656 12.9499 0.0344 2.17 1.089

35.0 1.7347 0.9815 10.1139 0.0185 1.89 1.096

40.0 1.7236 0.9869 8.7627 0.0131 1.82 1.079

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3.2.4. Viscosity measurements

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The viscosity experiment is believed to be one of the most unambiguous methods in clarifying of

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binding mode of a small molecule to the macromolecular architecture of ct-DNA. A classical

intercalative binding result in elongation of ct-DNA double helix associated with a significant
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rise in viscosity [47], whereas non-intercalative interaction such as coulombic and grooved mode

generally has no obvious effect on viscosity [48]. The effects of the complex and a typical
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intercalator, EB on the viscosity of ct-DNA are shown in Fig 10. It is observed that the viscosity
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of ct-DNA increases gradually with increasing the concentration ratio of EB, and this result is in
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line with the literature report [47]. However, the [Fe(pmaq)2]2+ complex leads to increase the
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relative specific viscosity of ct-DNA but to a considerably lower extent as compared

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to EB implying the existence of partial intercalation and/or minor groove binding interaction

between them (Fig 10).

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Fig.10. Effect of increasing amount of EB and [Fe(pmaq)2]2+ complex on the relative viscosity of
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ct-DNA at 298 K in 10 mM tris-HCl buffer, [ct-DNA]0 = 500 µM.

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3.2.5. Circular Dichroism studies

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Circular Dichroism spectral technique is a sensitive tool for diagnosing the changes in DNA
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morphology due to complex-DNA interaction. The changes of CD spectra of ct-DNA upon the

treatment of the [Fe(pmaq)2]2+ complex are shown in Fig.11. It is worth to note here that the

complex does not contribute to the CD spectra in the wavelength range of 220-330 nm. Hence in

Fig. 11, the CD spectra is believed to be exhibited exclusively by ct-DNA and characterized by

two distinct bands in the UV region, a positive band at 275 nm due to base stacking and a

negative band at 245 nm due to right handed B-form helicity [49]. Simple groove binding and

coulombic interaction of small molecules with DNA do not show any perturbation on the helicity

or base stacking of DNA whereas classical intercalators induce alterations of the band intensity
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due to stabilization of helicity and simultaneous increment of base stacking [50]. In our present

study, as we progressively increase the complex concentration, ellipticity of the positive band

increases considerably associated with a red shift of the band maxima while that of the negative

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band shifts towards zero level (Fig.11). The small bathochromic shift of the positive band by 3

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nm provides an important support to the partial intercalative mode of binding interaction of the

complex with ct-DNA.

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Fig. 11. CD spectra of ct-DNA (25 µM) in absence and presence of metal complex, where (i) →

(iv) correspond to 0.0, 5.0, 10.0 and 15.0 µM complex. The arrow indicates the change of

intensity with increasing complex concentration.

3.3. Molecular docking analysis

Molecular docking becomes an interesting and encouraging tool in recent years for

understanding the binding interaction of a synthesized compounds and biological

macromolecules. In our present work, molecular docking studies have been performed to find
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out the mode of binding of the synthesized [Fe(pmaq)2]2+ complex with most probable binding

sites of BSA and ct-DNA

3.3.1. Molecular docking with BSA

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It has been well established that BSA can bind with complexes principally through hydrophobic

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cavities located in sub-domain IIA and IIIA, which exhibit similar chemical properties [51].

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According to Sudlow et al, the binding cavities in sub-domain IIA and IIIA are also referred to

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as site I and site II respectively [52]. In site I, Trp-213 is known to be housed, while Tyr residue

is located in site II [53]. In order to substantiate the experimental results regarding BSA–
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[Fe(pmaq)2]2+ interaction, 25 possible docked conformations of BSA–[Fe(pmaq)2]2+ adduct have

been modeled by AutoDock program (as mentioned in the method section), out of which the
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conformation with the highest negative binding energy has been considered to be the best ranked
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results, and is shown in Fig. 12. The binding energy of the most stable docked form (Fig. 12a) is
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evaluated as –21.12 kJ/mole. However the difference in binding energy values computed
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theoretically (21.12 kJ/mol) and that calculated experimentally ((33.55±0.81)kJ/mol) lies in the

fact that the structure from X-ray crystallography of BSA is significantly different from that in
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aqueous system used in our present study. The molecular docking studies indicate that the

complex, [Fe(pmaq)2]2+, is in close proximity to the hydrophobic residues of site II at sub-

domain IIIA (Fig. 12a). The close-up view of this docked form is shown in Fig. 12b which

reveals that [Fe(pmaq)2]2+is surrounded by a number of amino acid residues, namely,Tyr-496,

Ala-500, Lys-499, Pro-498, Val-497 and Gln-416 (Fig. 12b). Hence, the results obtained from

the above docking studies implies that the interaction between [Fe(pmaq)2]2+ and BSA is

dominated by hydrophobic forces and this is in consistent with the spectroscopic findings that we

have already gathered.

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Fig. 12: (a) is the lowest energy binding mode of [Fe(pmaq)2]2+ to BSA. The secondary structure
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of BSA is displayed by ribbon and tube, and [Fe(pmaq)2]2+is displayed by space ball and

coloured in magenta. (b) is the close-up view of binding site of [Fe(pmaq)2]2+ on BSA
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corresponding to (a), and the selected amino acid residues are shown by stick model.

3.3.2. Molecular docking with ct-DNA

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The biological activity of a compound can be better understood from the knowledge of its

binding location in ct-DNA architecture [39]. Thus, a molecular docking study has been

accomplished herein to find out the binding site of [Fe(pmaq)2]2+ in ct-DNA (B-form with pdb

id: 1BNA) along with preferred orientation of the complex inside the ct-DNA grooves. Out of 25

possible docked forms, the energetically most favorable conformation of [Fe(pmaq)2]2+ is shown

in Fig. 13a, which reveals that [Fe(pmaq)2]2+ complex binds to the minor groove of the ct-DNA

The close-up view of this docked form is presented in Fig. 13b. The ct-DNA bases (orange

coloured) present within a distance of 4 Å around the complex (Fig. 13b) indicates that the
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complex bind to ct-DNA towards the G-C rich region due to van der waals interaction and

hydrophobic contacts with the functional groups of DNA. The binding energy of the most stable

docked form has been found to be 23.1 kJ/mol. The observation from docking study is in line

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with the results obtained from spectroscopic and viscometric techniques which provide important

information on the mode of binding of [Fe(pmaq)2]2+ complex to ct-DNA.

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Fig. 13: (a) Lowest energy docked form of [Fe(pmaq)2]2+ complex with ct-DNA. The structure of
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ct-DNA (cyan coloured) and [Fe(pmaq)2]2+complex (magenta coloured) are displayed ball and

stick model, (b) is the close-up view of binding site of [Fe(pmaq)2]2+ on ct-DNA corresponding

to (a). The orange coloured residues are within a distance of 4 Å around the complex at the

interaction site.

4. Conclusion
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Synthesized Schiff base Fe(II) complex, [Fe(pmaq)2]2+ shows noticeable binding propensity with

bio-molecules BSA and ct-DNA. Steady-state and time-resolved fluorescence measurements

reveal that both static and dynamic quenching are operating in reducing the BSA emission

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intensity by the Fe(II) complex. CD studies show that α-helicity of BSA decreases from 59.4%

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(native state) to 56.8% implying secondary structural alteration of BSA induced by the complex.

However a number of spectroscopic techniques coupled with viscometric technique reveal partial

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intercalative binding interaction of the complex with ct-DNA. Molecular docking analysis shows

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that [Fe(pmaq)2]2+ binds near the hydrophobic residues of site II of BSA at sub-domain IIIA

while the docked conformation of [Fe(pmaq)2]2+ with ct-DNA reveals that the complex bind
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towards the G-C rich region of ct-DNA. Thus the knowledge gained from this study can be

employed for the development of potential probe for BSA and ct-DNA structure.
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Acknowledgement
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SR is grateful to the Department of Science & Technology (DST), New Delhi, for INSPIRE
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fellowship and SD & CP sincerely acknowledge University Grants Commission (UGC), New
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Delhi, for senior research fellowship.

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